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* This work was supported, in whole or in part, by National Institutes of Health Grants DA026947, DA021471, and NS071122. The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. 1–3.
Dysregulation of dopamine (DA) homeostasis is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. α-Synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. To elucidate the potential functional consequences of DAT/α-synuclein interaction, we explored α-synuclein modulation of DAT activity in midbrain dopaminergic neurons obtained from TH::RFP mice, immortalized DA neurons, and a heterologous system expressing DAT. We used dual pipette whole cell patch clamp recording to measure the DAT-mediated current before and after dialysis of recombinant α-synuclein into immortalized DA neurons. Our data suggest that intracellular α-synuclein induces a Na+ independent but Cl−-sensitive inward current in DAT-expressing cells. This current is blocked by DAT blocker GBR12935 and is absent when heat-inactivated α-synuclein is dialyzed into these cells. The functional consequence of this interaction on DAT activity was further examined with real-time monitoring of transport function using a fluorescent substrate of DAT, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+). Overexpression of α-synuclein in DAT-positive immortalized DA neurons and CHO cells expressing DAT decreased the magnitude and rate of DAT-mediated substrate uptake without a decrease in the initial binding of the substrate at the plasma membrane. Taken together our findings are consistent with the interpretation that DAT/α-synuclein interaction at the cell surface results in a DAT-dependent, Na+-insensitive, Cl-sensitive inward current with a decrease in substrate uptake, suggesting that DAT/α-synuclein interaction can modulate dopamine transmission and thus neuronal function.
The development of effective therapeutic interventions for neurodegeneration requires a better understanding of the early events that precede neuronal loss. Recent work in various disease models has begun to emphasize the significance of presynaptic dysfunction as an early event that occurs before manifestation of neurological disorders (
). By removing extracellular DA and recycling it back into the neuron, DAT plays an essential role in the replenishment of synaptic DA and in terminating DA signaling. An increasing number of proteins have been identified that interact with different domains of the dopamine transporter. These interactions suggest that the synaptic distribution and functional properties of DAT can be regulated via interacting proteins. Most of the known DAT-interacting proteins have been identified using the yeast two-hybrid system (Y2H). The DAT-interacting proteins identified to date using the Y2H system include protein that interacts with C kinase-1 (PICK1) (
Abnormal accumulation of α-synuclein is strongly implicated in Parkinson disease. For example, massive accumulation of α-synuclein in the substantia nigra of patients with triplication of α-synuclein and in midbrain dopamine neurons of chronic cocaine abusers are reported (
) reported that high concentrations of α-synuclein (more that 1700% of control) are found in neurodegenerative disorders such as Parkinson disease, familial Parkinsonism with dementia, and multiple system atrophy. Similarly, the Mash laboratory (
The role of α-synuclein in dopaminergic transmission and its underlying mechanism is not fully understood, primarily because data to date appear conflicting. Although Lee et al. have shown that α-synuclein overexpression results in DAT clustering at the plasma membrane and increased DAT function (
) have reported a decreased cell surface expression of DAT and inhibition of DAT activity when α-synuclein is overexpressed.
To address these conflicting findings and further explore the functional consequences of DAT/α-synuclein interaction at the cell surface, we utilized single cell dual pipette whole cell patch clamp recordings to measure DAT-mediated current before and after dialysis of recombinant α-synuclein into immortalized DA neurons expressing dopamine transporter. We found that introducing α-synuclein intracellularly induces a Na+-independent but Cl−-sensitive inward current in immortalized DA neurons and DAT-expressing cells, which is eliminated by the DAT antagonist GBR12935 and is absent when the cell is dialyzed with heat-inactivated α-synuclein. Furthermore, our finding suggests that overexpression of α-synuclein decreases DAT-mediated substrate uptake without decreasing the overall number of transporters at the membrane.
Reagents and drugs were purchased from Sigma unless otherwise noted. α-Synuclein was purchased from Millipore (Temecula, CA). α-Synuclein cDNA plasmid was a generous gift from Dr. Ted M. Dawson, Johns Hopkins School of Medicine.
All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals. Mouse midbrain neuronal cultures were obtained from a transgenic mouse strain generated as described previously (
). Briefly, the tyrosine hydroxylase promoter-driven red fluorescent protein transgene (TH::RFP) was constructed by ligating a 4.5-kb HindIII/EcoRI fragment of the rat tyrosine hydroxylase promoter with DsRed2–1 (Clontech, Palo Alto, CA). The acutely dissociated mouse midbrain dopaminergic neurons from 1–3-day-old pups were isolated and grown on a monolayer of glial cells on poly-d-lysine-treated glass-bottom dishes as we have described previously (
Immortalized DA Neuronal Culture, Cell Lines, and Cell Culture
The immortalized DA neurons 1RB3AN27 cells or 1RB3AN27 cells overexpressing human DAT used in this study were gifts from Dr. Haley Melikian (University of Massachusetts) and have been characterized previously (
). Cells double every 26 h, which upon differentiation contain tyrosine hydroxylase and DAT proteins and their respective mRNAs. If untreated, the undifferentiated neurons only have the potential to express DAT. For electrophysiology experiments we used undifferentiated immortalized DA neurons (1RB3AN27 cells) and undifferentiated immortalized DA neurons overexpressing human DAT. Immunocytochemistry experiments have confirmed previous reports that undifferentiated immortalized DA neurons do not express DAT unless they are engineered to overexpress DAT (data not shown). Cells were grown in RPMI medium supplemented with 5% glutamine, 10% fetal bovine serum at 37 °C, and 5% CO2. To keep the DAT-expressing cells under selection, 1 μl/ml G14 was added to the media. For imaging experiments, where we needed a fluorophore-transporter fusion protein to track the transporter in real time, we used parental or stably over expressing YFP-DAT CHO cell lines. The CHO cell lines were gifts from Dr. Jonathan Javitch (Colombia University). Previous studies have shown that these cells are flat, have minimal basal fluorescence, and are suitable for image analysis (
). The cells were grown in F12 medium supplemented with 10% fetal bovine serum, 5% glutamine at 37 °C and 5% CO2, and 1.5 μl/ml hygromycin.
Subcloning of α-Synuclein into Enhanced Cerulean Fluorescent Protein (CFP) Expression Vector
The cDNA encoding α-synuclein was subcloned into enhanced CFP expression vector (a generous gift from Dr. David Piston, Vanderbilt University), using PCR and GGTGAACTTTCATGGATGTATTCATGAAAGGA (upstream) and GCGT CTAGAGGCTTCAGGTTCGTAGTC (downstream). The PCR product was digested with HindIII and XbaI and ligated into multiple cloning sites of pRC/CMV vector. The ligation product was transformed into Escherichia coli strain DH5α, and the cDNA was purified and successfully subcloned. cDNA was purified in large scale using Maxi-prep kit (Qiagen, Valencia, CA). Sequencing confirmed the product obtained was α-synuclein.
Western Blot Analysis
Western blot analyses were performed as described previously (
). In brief, YFP-DAT-CHO cells, IRBN27-DAT cells, or midbrain samples were washed with ice-cold 0.1 m PBS, homogenized in lysis buffer (0.01 m Tris-HCl, pH 7.6, 0.1 m NaCl, 0.001 m EDTA, 100 μg/ml PMSF, and 1 μg/ml aprotinin), and then centrifuged at 15000 × g for 30 min. Protein concentrations were determined using the Bio-Rad D Protein assay (Bio-Rad). 20 μg of total protein was electrophoresed on 10% SDS-polyacrylamide gels and then transferred to Hybond-P™ membranes (GE Healthcare) as described previously (
). The membranes were blocked and then incubated with a primary antibody directed against α-synuclein (1:1000) (Millipore, Billerica, MA) at 4 °C overnight. Immune complexes were detected with an HRP-labeled secondary antibody and ECL+ chemiluminescence reagents (GE Healthcare). To confirm equal protein loading, blots were stripped and reprobed with an anti-α-actin antibody (1:3000; Sigma) for 2 h at room temperature. Signal intensity was measured using densitometric analysis and quantified.
Measurement of DAT-mediated Whole Cell Currents
Cells were plated at 105/35-mm culture dish. Attached cells were washed three times with external solution at room temperature. The external solution contained 130 mm NaCl, 10 mm HEPES, 34 mm dextrose, 1.5 mm CaCl2, 0.5 mm MgSO4, and 1.3 mm KH2PO4 adjusted to pH 7.35 with a final osmolarity of 290 mosm. The pipette solution for the whole cell recording contained physiological-like internal solution containing 120 mm CsCl, 0.1 mm CaCl2, 2 mm MgCl2, 1.1 mm EGTA, 10 mm Hepes, and 30 mm dextrose. The dialysis solution contained physiological-like internal solution ±5 μm α-synuclein or heat-inactivated α-synuclein (as specified in the “Materials and Methods” and “Results” sections). In ion substitution strategies we used either iso-osmotic substitution of choline for Na+ in the external solution to completely remove extracellular Na+ or we used partial substitution of extracellular chloride for sodium acetate to increase Cl− gradient from 133 to 90 mm (i.e. extracellular Cl− concentration decreased to 90 mm). Thus, in all of the experiments, the intracellular Cl− concentration is at constant levels of Cl− = 124.2 mm. pH and osmolarity of the internal solutions were kept constant at 7.35 and 270 mosm, respectively.
Patch electrodes were pulled from quartz pipettes on a P-2000 puller (Sutter Instruments, Novato, CA) and filled with the pipette solution. Whole cell currents were recorded using an Axopatch 200B (Molecular Devices, Sunnyvale, CA) with a low-pass Bessel filter set at 1000 Hz. Current-voltage relations were generated using a voltage step (500 ms) protocol ranging from −100 to +60 mV separated by 20 mV from a holding potential of −20 mV. Data were recorded and analyzed off-line using pCLAMP9 software (Molecular Devices).
We used a technique we term “dual whole cell patch clamp” electrophysiology, a modification of a recording configuration used by Mazzanti and DeFelice (
) to determine the effect of α-synuclein on DAT-mediated whole cell currents in immortalized DA neurons stably overexpressing DAT. The dual whole cell patch clamp electrophysiology was performed as follows; one electrode is designated as the recording electrode, and the second electrode is designated as the dialysis electrode. 1) Two gigaohm seals are acquired with two electrodes on the same cell. 2) Whole cell voltage clamp mode is achieved in the “recording” electrode, and 10 min later an I(V) curve is measured. 3) Whole cell current clamp is achieved in the “dialysis” electrode. We briefly placed the recording electrode in current clamp mode, whereas the membrane test voltage verified break-in of the dialysis electrode. Once the dialysis electrode has achieved whole cell configuration, it is placed in current clamp mode, and the recording electrode is returned to voltage clamp mode. Our laboratory and other laboratories have demonstrated that maximum dialysis occurs within 8–10 min after establishing the whole cell configuration (
). The cell is thereby dialyzed with the contents of the dialysis electrode for 10 min followed by an I(V) recording (Fig. 1) (using command potentials generated by the voltage clamp recording electrode). As a control, the voltage of the dialysis electrode was monitored to assure the recording electrode had a tight voltage clamp throughout the experiment. The dual whole cell technique was performed with the recording and dialysis electrodes both containing internal solution (
), as described (Fig. 1), or the recording electrode filled with internal solution and the dialysis electrode containing internal solution +5 μm α-synuclein or heat-inactivated α-synuclein (Fig. 2). The dual whole cell experimental configuration permits measurement of I(V) before and after α-synuclein dialysis. The external solution contained either external solution or contained an iso-osmotic ion substitution when ion substitution strategies were conducted. In this document consistent with electrophysiological nomenclature, an “inward” current refers to an ionic event that requires the clamp electrode to pass negative current into the cell to keep the voltage clamped at a given potential. As such, the term inward in this context does not refer to the direction of the ion flow; hence, the flow of positive ions into the cell and/or flow of negative ions out of the cell both produce an inward current. In this study the DAT-dependent current is defined as currents we measure, based on our previous studies (
), that are blocked by DAT blockers, like GBR12935.
Selection of the 5 μm Concentration of α-Synuclein for Use in Our Recording Pipette
We used quantitative or semiquantitative neurochemical measurements of the full-length 19-kDa α-synuclein that was extrapolated against a standard curve calibrated by using 1) purified recombinant α-synuclein, 2) cells overexpressing α-synuclein, and 3) α-synuclein levels in the midbrain region of animals treated with neurotoxic doses of methamphetamine (an animal model of system atrophy).
J. Gamble-George and H. Khoshbouei, unpublished data.
These criteria suggest that 5 μm recombinant α-synuclein (approximately 0.07 μg of recombinant human α-synuclein) can be detected as a 19-kDa protein (supplemental Fig. 2) and is in the median range of our quantitative or semiquantitative neurochemical measurements. Therefore, in a reduced system such as ours, 5 μm α-synuclein in the recording pipette may actually be an underestimation of actual increase in α-synuclein levels reported in synucleinopathies (see the Introduction), (
). For ASP+ experiments, cells were plated at 105/35-mm glass-bottom MatTek Petri dish (MatTek, Ashland, MA). CHO or immortalized DA neurons cells stably expressing YFP-DAT were transiently transfected with CFP-α-synuclein. The DAT-mediated ASP+ uptake was similar in either cell lines. Images of CHO-YFP-DAT cells are shown because these cells provide better visualization of the data. The addition of YFP tag to DAT does not alter transporter-mediated activity (
). The cells were washed 2× with external solution with a final pH of 7.35 and osmolarity of 290 mosm as described above. 1 ml of external solution was added to the dish, and cells were selected for imaging with a Nikon inverted TE2000-U C1 laser scanning confocal microscope (Nikon, Melville, NY). A 40 × 1.3 NA Plan-fluor objective (Nikon) was used with the confocal pinhole set to one Airy unit. Pilot experiments at both room temperature and 37 °C were performed; the relative difference between experimental conditions was unchanged regardless of temperature (data not shown); however, to be comparable with the preponderance of the ASP+ literature (
), we chose to perform our experiments at room temperature. The cells were selected with the appropriate filter sets. The CFP-α-synuclein was identified using a CFP-specific filter set, and YFP-DAT was identified using a YFP filter set. For analysis purposes, at the start of each experiment 512 × 512 pixel reference images showing cells expressing YFP-DAT alone, cells co-expressing CFP-α-synuclein/YFP-DAT, cell expressing CFP-α-synuclein alone, or cells expressing neither protein were saved and labeled for analysis. This provides an internal experimental control because not every cell co-expresses both proteins. Using this approach, real time quantification of DAT-mediated substrate uptake in single cells was measured using the high affinity, fluorescent DAT substrate ASP+ (
). The sampling frequency was 1.52 frame per second (fps) over 8 min that provided 692 images. After 10 base-line images, ASP+ was added to a final concentration as noted under “Results” (FIGURE 5, FIGURE 6). ASP+ was excited at 488 nm and detected at 590/50 nm. The emission of YFP signal is significantly blue-shifted compared with ASP+ maximum emission that minimizes bleed-through into the ASP+ channel. At the start of each experiment, the detector gain in the ASP+ channel was adjusted to offset any base-line YFP bleed-through (
Data were pooled from at least three separate transfections, with a minimum of four independent experiments per transfection. Cell boundaries were determined from an image showing both YFP intensity and differential interference contrast images saved before the timed experiment. Binding and transport of ASP+ was determined from the average fluorescent units of each individual cell throughout each experiment using the NIS elements software package (Nikon). Within each experiment three groups of cells were selected for analysis: YFP-DAT/CFP-α-synuclein, YFP-DAT/only, and cells expressing neither protein. This approach provided an internal control resulting in the evaluation of the two experimental groups under identical conditions. Fluorescence measurements were normalized by dividing the average of the first four fluorescence values. Values are shown as the fold change in ASP+ fluorescence, and the rate was defined as the slope of ASP+ uptake (
) (from a linear fit line from the data at t = 75–250 s).
Choice of Cells for Various Experiments
For electrophysiological studies we used immortalized DA neurons, i.e. 1RB3AN27 cells overexpressing human DAT. The results were confirmed in CHO-YFP-DAT cells and midbrain DA neurons obtained from primary neuronal culture of acutely dissociated TH::RFP mouse midbrain dopamine neurons. For control experiments we used relevant parental cells not expressing DAT.
For DAT-mediated substrate (ASP+) uptake studies, we used immortalized DA 1RB3AN27 neuronal cells overexpressing DAT. All of the uptake experiments were confirmed in CHO-YFP-DAT cells. We have shown images of CHO-DAT cells because these cells have low background fluorescence and thus less confounding visual representations of the data. We were not able to use primary cultures from midbrain DA neurons of TH::RFP mouse for the uptake experiments because the high endogenous fluorescence of these cells confounded detection of ASP+.
Statistical Analyses Performed
Data are shown as the mean ± S.E. Unless otherwise noted, analysis of variance with post hoc Newman-Keuls was used for the statistical comparison of more than two means. The two-tailed, unpaired Student's t test was applied when two means were compared.
Data are shown as the mean ± S.E. unless otherwise noted. In ASP+ competition experiments, the data were fit with GraphPad Software Prism 5.0 using the least squares method to fit a regression line (at t = 75–250 s). (Fig. 6A). Data were analyzed for rejection of the null hypothesis using a one-way analysis of variance followed by a Bonferroni post hoc test (FIGURE 5, FIGURE 6; supplemental Tables 1 and 2). Tests were performed using Prism Version 5.0 statistical software package (GraphPad Software). A value of p < 0.05 was considered statistically significant. F values, degrees of freedom, and significance are also documented in supplemental Tables 1 and 2.
Regulation of DAT activity plays a key role in extracellular dopamine availability. The findings that DAT interacts with α-synuclein (
), a protein implicated in neurodegenerative disease, provoked our interest in functional consequence of this interaction on DAT activity. High concentrations of α-synuclein (more than 1700% of control level) are found in neurodegenerative disorders such as Parkinson disease, familial Parkinsonism dementia, and multiple system atrophy (
). Using the single cell, dual patch clamp approach, we were able to focus on the impact of α-synuclein on DAT activity in real time. We found that intracellular α-synuclein induces a DAT-mediated inward current that is independent of extracellular Na+ but sensitive to the transmembrane Cl− gradient. This inward current is eliminated by DAT antagonist GBR12935 and is absent with intracellular heat-inactivated α-synuclein. Intracellular α-synuclein mediates a rightward shift in the DAT reversal potential and depolarizes the membrane. These changes are paralleled by an α-synuclein-dependent decrease in rate of DAT-mediated substrate uptake.
Our studies directly assess the effect of α-synuclein on DAT activity when α-synuclein is dialyzed into DA neuron (
) via the patch clamp electrode or when α-synuclein is transiently overexpressed. Dialysis of α-synuclein into the cell via the patch clamp electrode allows real time assessment of DAT activity before and after intracellular α-synuclein under identical conditions and, importantly, in the same cell. Therefore, it provides a unique experimental configuration that can assess the influence of intracellular α-synuclein on the activity of the transporter in a voltage-controlled condition.
DAT-associated currents are reported to arise from the stoichiometric coupling of two Na+ ions transported and one Cl− ion counter-transported to each molecule of substrate transported (
) have shown that DAT-mediated currents can occur in the absence of the substrate and are larger than the predicted stoichiometry of the transport cycle. Therefore, it is postulated that DAT-associated current can be characterized as both coupled and uncoupled currents via both channel and transporter modes of DAT operations (
). Consistent with the literature, we found that dialysis of α-synuclein into the cell via the patch clamp electrode results in a DAT-mediated uncoupled inward current that is eliminated by DAT blocker GBR12935. Intracellular α-synuclein mediates a rightward shift in the DAT reversal potential that may be attributed to increases in the inward flow of Na+ and/or outward flow of Cl− ions (see below). Consistent with this observation, we found that the membrane potential of DAT cells overexpressing α-synuclein rest at more depolarized membrane potentials (Fig. 2).
We are not the first group to report an increase in the DAT-mediated uncoupled currents (
) previously observed that in the absence of extracellular DA, arachidonic acid (10–100 μm) stimulates a cocaine (DAT blocker)-sensitive, DAT-associated inward current that is 50-fold greater than the inward current normally measured in DAT- expressing oocytes.
We also observe that α-synuclein overexpression depolarizes the membrane potentials from −36.5 ± 5.5 mV in DAT cells and −20.3 ± 3.2 mV in DAT/α-synuclein cells (p < 0.05) (Fig. 2B). This α-synuclein-regulated change in the resting membrane potential can affect both the excitability of the cell and its energy expenditure. Thus, α-synuclein interaction with DAT may affect neuronal energy consumption that could lead to disruption of cell homeostasis. Because α-synuclein is implicated in neurodegenerative diseases, examination of the metabolic cost of α-synuclein overexpression in dopamine neurons is of interest.
One of our goals in this study was to possibly shed some light on the conflicting data concerning the functional consequences of DAT/α-synuclein interaction. Our data are consistent with previous reports by Wersinger et al. (
), also examining [3H]DA uptake in cell populations, see increases in DA when α-synuclein is overexpressed. However, in neither of these [3H]DA uptake studies was it possible to visualize DAT at the cell surface or measure live cell instantaneous (millisecond) rates of substrate at the single cell level. It is possible that the impact of α-synuclein on DAT-mediated DA efflux, DAT trafficking, or other events that are asynchronous in their multicell studies limit the interpretation of steady state measurement of [3H]DA uptake in the cell population studies.
The magnitude and rate of dopamine uptake and dopamine efflux is determined not only by the number of transporters at the surface membrane but also by the conformational state of the transporters; that is, inward facing conformation versus outward facing conformation. These conformational states can be regulated by membrane potential (
). Because we found that total DAT protein (supplemental Fig. 1) and the maximum initial ASP+ binding between the two groups, DAT cells and DAT with and without α-synuclein cells, were similar (Fig. 5C), our data provide yet another level of molecular detail for the impact of α-synuclein on DAT function and are consistent with the interpretation that α-synuclein alters the ionic coupling of DAT to decrease DAT-mediated substrate uptake without any change in the surface expression of DAT.
The dopamine transporter has been shown to affect membrane excitability (
), which in turn can impact DAT properties. The data presented in this study cannot exclude these possibilities. However, the studies do affirm the importance of DAT in the changes in currents observed when α-synuclein is overexpressed, as these currents are not detected in the absence of DAT, when the transporter is blocked by an antagonist, or when heat inactivated rather than when native α-synuclein is introduced into the recording pipette.
The variety of ways in which α-synuclein expressed at normal endogenous levels regulates DAT activities is still being clarified. For example, α-synuclein may function as a co-chaperone, as transgenic expression of α-synuclein abolishes the lethality and neurodegeneration caused by deletion of the synaptic vesicle chaperone, cysteine-string protein-α (
). In addition, α-synuclein also has been suggested to be involved in intracellular trafficking, as overexpression of α-synuclein in Drosophila and yeast disrupts cargo trafficking within the endoplasmic reticulum (
), although studies in the context of various stressors in these mice have yet to be performed. Nonetheless, the distinctive neuropathology found in hereditary parkinsonism and dementia is linked to α-synuclein overexpression (
). Thus, our studies are important in revealing the molecular consequences of this increased α-synuclein expression.
Overall, however, our findings provide evidence that interaction of α-synuclein and surface DAT regulates the activity of the transporter that can modulate the synaptic availability of DA in addition to dramatically affecting the membrane microenvironment near the transporter, which may alter homeostasis of DA neurons. Together, these changes would alter multiple properties of DA terminals. The functional consequences of these biophysical changes to in vivo physiology and pathology are yet to be discovered.
Microscopy experiments were performed using the Meharry Medical College Morphology Core, which is supported in part by National Institutes of Health Grants U01NS041071, U54RR026140, and S10RR0254970. We thank Drs. Lee Limbird and Louis DeFelice for helpful suggestions and critical review of this manuscript.